Cellular-level phenotyping of tumor-immune microenvironment (TiME) in patients in vivo reveals distinct inflammation and endothelial anergy signatures

Immunotherapies have shown unprecedented clinical benefits in several malignancies[1][1]–[3][2]. However, clinical responses remain variable and unpredictable, indicating the need to develop predictive platforms that can improve patient stratification[4][3]. Phenotyping of tumors into hot, altered, or cold[5][4] based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response[6][5],[7][6]. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking[5][4],[8][7],[9][8] also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging[10][9], we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. Our novel in vivo cellular-level imaging and phenotyping approach can potentially advance our fundamental understanding of TiME, develop robust predictors for immunotherapy outcomes and identify novel targetable pathways in future. ### Competing Interest Statement Melissa Gill: consulting investigator for DBV technologies; research consultant: Dermatology Service, MSKCC. Christi Alessi-Fox: employee of and owns equity in Caliber I.D., manufacturer of the VivaScope RCM. Dr. Rossi: Mavig (travel accommodation), Merz, DynaMed, Canfield Scientific, Evolus, Biofrontera, QuantiaMD, Lam Therapeutics, Cutera (consultant); Allergan (advisory board). Allan Halpern: consultant to Canfield Scientific and an advisory board member of Scibase. L.D. is a cofounder and holds equity in IMVAQ Therapeutics. She has patents on applications related to work on oncolytic viral therapy. Ashfaq A. Marghoob: honorarium for dermoscopy lectures (3GEN), royalties for books/book chapters, dermoscopy equipment for testing, payment for organizing and lecturing (American Dermoscopy Meeting). Chih-Shan Jason Chen: research funding from Apollo Medical Optics, Inc. Milind Rajadhyaksha: was employee of and owns equity in Caliber I.D. VivaScope is the commercial version of a laboratory prototype he developed at Massachusetts General Hospital, Harvard Medical School. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-5 [5]: #ref-6 [6]: #ref-7 [7]: #ref-8 [8]: #ref-9 [9]: #ref-10